1. Field of the Invention
A new method relates to determining the functional activity of the MRP2 and/or MRP3 efflux pathway of a human or animal subject, and to the use of a bile acid derivative in such a method.
2. Background of the Invention
MRP2/Mrp2 (ABCC2/Abcc2), also known as Multidrug Resistance Protein 2, is a member of the ABCC subfamily of the ATP-Binding Cassette Transporter superfamily. An overview of the ATP-Binding Cassette transporter family is given in ‘The Human ATP-Binding Cassette (ABC) Transporter Superfamily’ by Michael Dean of the National Cancer Institute at Frederick, Md., published by the US National Center for Biotechnology Information. MRP2 is expressed on the apical domain of hepatocytes, enterocytes of the proximal small intestine, and proximal renal tubular cells, as well as in the brain and the placenta.
MRP2 is important clinically as it modulates the pharmacokinetics of many drugs, and its expression and activity are also altered by a range of drugs and diseases. For example, expression of MRP2 is altered in patients with liver diseases such as primary biliary cirrhosis, cholestasis (e.g. drug-induced cholestasis), fatty liver disease, alcoholic liver disease, primary sclerosing cholangitis, or viral hepatitis. In the kidney, MRP2 functions in renal elimination of substrates from the blood into urine. In the liver, MRP2 is the major exporter of organic anions from the liver into the bile. MRP2 can transport sulphated and glucuronidated bile salts as well as other organic anions and/or their conjugates with glucuronate, glutathione and sulphate [Gerk et al. J Pharmacol Exp Ther 2002 August; 302(2):407-415]. In addition to transport of conjugates, MRP2 transports a range of molecules including cancer chemotherapeutics, uricosurics, antibiotics, leukotrienes, glutathione, toxins and heavy metals. MRP2 also plays an important role in the elimination of bilirubin glucuronosides from hepatocytes into bile. The MRP2 gene is mutated in patients with Dubin-Johnson syndrome, a human disorder of the organic ion transport. The absence of functional MRP2 from the canalicular membrane causes conjugated hyperbilirubinemia, as observed in the hereditary disorder Dubin-Johnson syndrome.
Loss of MRP2 function is often well tolerated and compensated by the upregulation of other membrane transporters, particularly Multidrug Resistance Protein 3 (MRP3, also known as ABCC3) in the basolateral member of hepatocytes [Nies et al. Pflugers Arch (2007) 453:643-659]. Like MRP2, MRP3 is a member of the ABCC subfamily of the ATP-binding cassette transporter superfamily. MRP3 is present in the intestine, kidney and the liver.
Knowledge of the state of the MRP2/3 activity of a subject therefore could be used to indicate a wide range of possible conditions.
It would therefore be advantageous to provide a method of determining the functional activity of the MRP2 and/or MRP3 efflux pathway of a human or animal subject.
We have unexpectedly found that MRP2 is the most prominent transporter responsible for biliary excretion of cholyl lysyl fluorescein (CLF), which is also known as fluorescein lisicol, and that MRP3 is responsible for basolateral excretion of CLF. Accordingly, CLF and other bile acids having a similar metabolic/elimination pathway can be used in determining the functional activity of MRP2/3 efflux pathway.
Some aspect of the present invention is particularly surprising in view of the disclosure of Mills et al. in Biochim Biophys Acta 1991 Dec. 6; 1115(2):151-156. They showed that, in rats, the biliary excretion rate of CLF after jugular vein injection has similar kinetics to that of glycocholate, the transport of which is known to be mediated by ABCB11. ABCB11 is a member of the ABCB subfamily of the ATP-binding cassette (ABC) transporter superfamily.
Other evidence that led to the belief that CLF is transported via ABCB11 was provided by Baxter et al. [Biochim Biophys Acta 1995 Jun. 6; 1256(3):374-380]. Baxter et al. administered glycocholate and CLF to isolated perfused rat livers under recycling conditions, and observed that CLF was capable of increasing phospholipids and cholesterol output in a similar way to that which was found for glycocholate. It was shown in the same study that rat liver has a much greater capacity to transfer glycocholate (GC) from perfusate to bile than CLF and concomitantly, the increase in phospholipid and cholesterol output was less with CLF in comparison with GC.
In a later study [Mills et al. in J Hepatol 1999 October; 31(4):678-684], Mills et al. investigated ABCC2/Abcc2 as a possible transporter for CLF. But they concluded from a study with normal and TW (Abcc2 deficient) Wistar rats that Abcc2 was not involved in the biliary excretion of CLF, based on the observation of similar biliary excretion in both strains.
In summary, prior to the present investigations, the totality of the prior art on the subject concluded unanimously that CLF was transported from the liver into bile by the ABCB11/Abcb11 pathway, rather than the ABCC2/Abcc2 pathway, and therefore would not have been considered as an agent for use in a method for the determination of MRP2/3 activity.